Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases.
Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases.
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DOI：
10.1002/cbic.202100688
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发表时间：
2022-03-04
期刊：
影响因子：
3.2
通讯作者：
Scrutton, Nigel S.
中科院分区：
文献类型：
--
作者：
Leferink, Nicole G. H.;Escorcia, Andres M.;Ouwersloot, Bodi R.;Johanissen, Linus O.;Hay, Sam;van der Kamp, Marc W.;Scrutton, Nigel S.
单萜脱氢酶通常是混杂的酶，由于涉及活性碳阳离子的分支反应机制的性质，产生产物混合物而不是纯化合物。两种先前鉴定的细菌单萜脱氢酶，芳樟醇合酶（bLinS）和桉叶素合酶（bCinS），分别从香叶基二磷酸产生几乎纯的芳樟醇和桉叶素。我们使用了一种结合实验和计算的方法来确定参与细菌单萜类化合物合成的关键残基。Phe 77对bCinS活性至关重要，引导线性碳阳离子中间体形成环状α-松油基中间体;去除芳环导致仅产生无环产物的变体。计算化学证实了Phe 77在碳正离子稳定中的重要性。Phe 74、Phe 78和Phe 179参与维持bCinS中的活性位点形状，而对芳环没有特定作用。bLinS中的Phe 295和bCinS中的等价物Ala 301分别是芳樟醇和桉叶素形成所必需的。当Phe 295对碳阳离子中间体进行空间限制时，Ala 301对bCinS初始环化和活性至关重要。我们的多学科方法提供了独特的见解，了解活性位点中精心放置的氨基酸残基如何通过空间限制或通过阳离子-π相互作用提供稳定性来引导碳阳离子沿着特定路径。 萜烯脱氢酶中的活性位点残基各自在将活性碳阳离子中间体引导至最终产物中具有独特且精确的功能。在这里，我们使用了一个相结合的实验和计算的方法来解开几个活性位点残基的作用，在细菌的单萜类糖苷酶。这些残基通常不保守，即使在密切相关的酶证明了定制的活性位点的重要性，为每个萜烯产品。
Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α‐terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation‐π interactions. Active site residues in terpene synthases each have a unique and precise function in guiding the reactive carbocation intermediates to the final product. Here we used a combined experimental and computational approach to unravel the role of several active site residues in bacterial monoterpene synthases. These residues are often not conserved even in closely related enzymes demonstrating the importance of a tailored active site for each terpene product.
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影响因子：
2.9
作者：
Li R;Chou WK;Himmelberger JA;Litwin KM;Harris GG;Cane DE;Christianson DW
通讯作者：
Christianson DW
影响因子：
2.9
作者：
Blank PN;Barrow GH;Chou WKW;Duan L;Cane DE;Christianson DW
通讯作者：
Christianson DW
影响因子：
2.7
作者：
FUJISAKI, S;HARA, H;NISHINO, T
通讯作者：
NISHINO, T
影响因子：
4.3
作者：
Lapczynski, A.;Letizia, C. S.;Api, A. M.
通讯作者：
Api, A. M.
影响因子：
12.9
作者：
Leferink, Nicole G. H.;Ranaghan, Kara E.;Scrutton, Nigel S.
通讯作者：
Scrutton, Nigel S.